The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes

Introduction

Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.

Methods

We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.

Results

eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.

Conclusions

These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.     

Comparative performance of contact plates,electrostatic wipes,swabs and a novel sampling device for the detection of Staphylococcus aureus on environmental surfaces

Aims

To evaluate the performance of four sampling methods [contact plates, electrostatic wipes (wipe), swabs and a novel roller sampler] for recovery of Staphylococcus aureus from a stainless steel surface.

Methods and Results

Stainless steel test plates were inoculated with Staph. aureus, dried for 24 h and sampled using each of the four methods. Samples were either incubated directly (roller, contact plate) or processed using elution and membrane filtration (swab, wipe). Performance was assessed by calculating the apparent sampling efficiency (ASE), analytical sensitivity (Sn) and percentage of replications with positive growth. The wipe demonstrated the best performance across all inoculating concentrations (ASE_48 h = 18%; Sn_48 h = 7 CFU per 100 cm²). The swab performed well when corrected for area actually sampled (ASE_48 h = 24%; Sn_48 h = 76 CFU per 100 cm²). Of the contact‐based methods, the newly developed roller sampler outperformed the contact plate (roller: ASE_48 h = 10%; Sn_48 h = 17 CFU per 100 cm²; contact plate: ASE_48 h = 0·04%; Sn_48 h = 1412 CFU per 100 cm²); both contact samplers performed better at higher inoculating concentrations (6E3 CFU per 100 cm² for the roller and 6E6 CFU per 100 cm² for the contact plate). Overall, the electrostatic wipe produced the highest number of replications resulting in positive growth (74%_24 h, 91%_48 h).

Conclusions

This study demonstrates that selection of the sampling method must be carefully considered, given that different methods have varying performance.

Significance and Impact of the Study

This is the first study assessing static wipes for sampling and one that uses a more real‐world‐relevant 24‐h drying time. The results help with infection control, and environmental health professionals choose better sampling methodologies.     

Understanding the placebo effect from an evolutionary perspective

A placebo is a treatment which is not effective through its direct action on the body, but works because of its effect on the patient's beliefs. From an evolutionary perspective, it is initially puzzling why, if people are capable of recovering, they need a placebo to do so. Based on an argument put forward by Humphrey [Great expectations: the evolutionary psychology of faith-healing and the placebo effect. In: Humphrey, N (2002). The mind made flesh. Oxford University Press, Oxford. 255–285], we present simple mathematical models of the placebo effect that involve a trade-off between the costs and benefits of allocating resources to a current problem. These models show why the effect occurs and how its magnitude and timing can depend on different factors. We identify a particular aspect of belief which may govern the effect and conclude that a deeper understanding of why the placebo effect exists may allow it to be invoked more easily in the future.     

Taxonomy and phylogeny of Puccinia lagenophorae: a study using rDNA sequence data, morphological and host range features

Puccinia lagenophorae is a rust fungus originating from Australasia which has spread throughout the world. A phylogenetic analysis of taxa related to this species was performed using rDNA (LSU, ITS) sequence data. The analyses revealed a well-supported cluster including all specimens of P. lagenophorae. By evaluating morphological and sequence data, the species is taxonomically re-defined and a list of synonyms is provided. Puccinia distincta on Bellis perennis, a species recently separated from P. lagenophorae, P. saccardoi, a species on the Goodeniaceae, and P. byliana, a species so far only known from South Africa, are reduced to synonymy in P. lagenophorae, as are several other species. Our analysis indicates that P. lagenophorae is likely not derived from the northern hemisphere species P. obscura, but from a species from Australia host-alternating between Asteraceae (aecial host) and Cyperaceae/Juncaceae (telial host). Another related species, P. stylidii (on Stylidium sp., Stylidiaceae) may have been derived from the same parental species as P. lagenophorae. From ontogenetical and morphological studies, the presence of pycnia could not be confirmed in the life cycle of this species, and the width of the pedicel of teliospores at the point of attachment was found to be highly variable and not a taxonomic character. The number of known host species is approximately 150, including 41 new host plants recorded herein.     

Suppression of inflammation by helminths: a role for the gut microbiota?

Multiple recent investigations have highlighted the promise of helminth-based therapies for the treatment of inflammatory disorders of the intestinal tract of humans, including inflammatory bowel disease and coeliac disease. However, the mechanisms by which helminths regulate immune responses, leading to the amelioration of symptoms of chronic inflammation are unknown. Given the pivotal roles of the intestinal microbiota in the pathogenesis of these disorders, it has been hypothesized that helminth-induced modifications of the gut commensal flora may be responsible for the therapeutic properties of gastrointestinal parasites. In this article, we review recent progress in the elucidation of host–parasite–microbiota interactions in both animal models of chronic inflammation and humans, and provide a working hypothesis of the role of the gut microbiota in helminth-induced suppression of inflammation.     

Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling

Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca²⁺ homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1^−/− mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca²⁺ entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca²⁺ entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca²⁺ entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit.     

Detection of virulence‐associated genes characteristic of intestinal Escherichia coli pathotypes,including the enterohemorrhagic/enteroaggregative O104:H4, in bovines from Germany and Spain

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real‐time PCR assays for the direct detection and quantification of nine virulence‐associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype‐related genes (wzx_O104, fliC_H4, rbf_O157, fliC_H7) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbf_O157/fliC_H7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzx_O104/fliC_H4) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzx_O104/fliC_H4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly‐virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early‐warning tool for the emergence of zoonotic E. coli in livestock.     

Protein phosphatase 2A regulatory subunits affecting plant innate immunity,energy metabolism,and flowering time – joint functions among B'η subfamily members

Protein phosphatase 2A (PP2A) is a heterotrimeric complex comprising a catalytic, scaffolding, and regulatory subunit. The regulatory subunits are essential for substrate specificity and localization of the complex and are classified into B/B55, B'', and B” non-related families in higher plants. In Arabidopsis thaliana, the close paralogs B''η, B''θ, B''γ, and B''ζ were further classified into a subfamily of B'' called B''η. Here we present results that consolidate the evidence for a role of the B''η subfamily in regulation of innate immunity, energy metabolism and flowering time. Proliferation of the virulent Pseudomonas syringae in B''θ knockout mutant decreased in comparison with wild type plants. Additionally, B''θ knockout plants were delayed in flowering, and this phenotype was supported by high expression of FLC (FLOWERING LOCUS C). B''ζ knockout seedlings showed growth retardation on sucrose-free medium, indicating a role for B''ζ in energy metabolism. This work provides insight into functions of the B''η subfamily members, highlighting their regulation of shared physiological traits while localizing to distinct cellular compartments.     

Spider Males Adjust Mate Choice but Not Sperm Allocation to Cues of a Rival

Spatial and temporal variation in ecological parameters selects for plastic responses to prevailing conditions. Males of web spiders have been shown to adjust developmental decisions to cues that relate to the degree of competition from other males. Here, we explore experimentally whether males of the spider Nephila senegalensis base behavioural decisions in the context of mate choice and sperm allocation to the presence of cues of a rival and to their own competitive dominance. Large and small males were offered a simultaneous choice between two penultimate instar females that varied in quality and in whether they were previously visited by a rival male. Large and small males avoided webs visited by a previous male, and this preference overrode differences in female quality. In a second experiment, males were assigned to copulate with a virgin female whose web was either previously visited by a rival male or not. We found substantial variation in male sperm allocation, but this variation was not related to our treatment. Our experiments demonstrate that males notice silk cues left behind by rival males on a female’s web and that they can utilize these cues in relation to decisions over whether to visit a web, but not in relation to sperm allocation.